ABSTRACT
Clinical, pharmacological, biochemical, and genetic evidence support the notion that alteration of cholesterol homeostasis strongly predisposes to Alzheimer disease (AD). The ATP-binding cassette transporter-2 (Abca2), which plays a role in intracellular sterol trafficking, has been genetically linked to AD. It is unclear how these two processes are related. Here we demonstrate that down-regulation of Abca2 in mammalian cells leads to decreased amyloid-ß (Aß) generation. In vitro studies revealed altered γ-secretase complex formation in Abca2 knock-out cells due to the altered levels, post-translational modification, and subcellular localization of Nicastrin. Reduced Abca2 levels in mammalian cells in vitro, in Drosophila melanogaster and in mice resulted in altered γ-secretase processing of APP, and thus Aß generation, without affecting Notch cleavage.
Subject(s)
ATP-Binding Cassette Transporters/biosynthesis , ATP-Binding Cassette Transporters/metabolism , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/metabolism , Drosophila Proteins/metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , ATP-Binding Cassette Transporters/genetics , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/genetics , Amyloid beta-Protein Precursor/genetics , Animals , Down-Regulation/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , HEK293 Cells , Humans , Membrane Glycoproteins/genetics , Mice , Nerve Tissue Proteins/genetics , RatsABSTRACT
Prodomains of A disintegrin and metalloproteinase (ADAM) metallopeptidases can act as highly specific intra- and intermolecular inhibitors of ADAM catalytic activity. The mouse ADAM9 prodomain (proA9; amino acids 24-204), expressed and characterized from Escherichia coli, is a competitive inhibitor of human ADAM9 catalytic/disintegrin domain with an overall inhibition constant of 280 ± 34 nM and high specificity toward ADAM9. In SY5Y neuroblastoma cells overexpressing amyloid precursor protein, proA9 treatment reduces the amount of endogenous ADAM10 enzyme in the medium while increasing membrane-bound ADAM10, as shown both by Western and activity assays with selective fluorescent peptide substrates using proteolytic activity matrix analysis. An increase in membrane-bound ADAM10 generates higher levels of soluble amyloid precursor protein α in the medium, whereas soluble amyloid precursor protein ß levels are decreased, demonstrating that inhibition of ADAM9 increases α-secretase activity on the cell membrane. Quantification of physiological ADAM10 substrates by a proteomic approach revealed that substrates, such as epidermal growth factor (EGF), HER2, osteoactivin, and CD40-ligand, are increased in the medium of BT474 breast tumor cells that were incubated with proA9, demonstrating that the regulation of ADAM10 by ADAM9 applies for many ADAM10 substrates. Taken together, our results demonstrate that ADAM10 activity is regulated by inhibition of ADAM9, and this regulation may be used to control shedding of amyloid precursor protein by enhancing α-secretase activity, a key regulatory step in the etiology of Alzheimer disease.
Subject(s)
ADAM Proteins/antagonists & inhibitors , ADAM Proteins/metabolism , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/metabolism , Cell Membrane/drug effects , Cell Membrane/enzymology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Peptide Fragments/pharmacology , ADAM Proteins/chemistry , ADAM10 Protein , Biocatalysis/drug effects , Cell Line, Tumor , Cell Membrane/metabolism , Humans , Membrane Proteins/chemistry , Protease Inhibitors/pharmacology , Protein Array Analysis , Protein Structure, TertiaryABSTRACT
The copper chaperone for superoxide dismutase (CCS) binds to both the ß-site AßPP cleaving enzyme (BACE1) and to the neuronal adaptor protein X11α. BACE1 initiates AßPP processing to produce the amyloid-ß (Aß) peptide deposited in the brains of Alzheimer's disease patients. X11α also interacts directly with AßPP to inhibit Aß production. However, whether CCS affects AßPP processing and Aß production is not known. Here we show that loss of CCS increases Aß production in both CCS knockout neurons and CCS siRNA-treated SHSY5Y cells and that this involves increased AßPP processing at the BACE1 site.
Subject(s)
Amyloid beta-Peptides/biosynthesis , Molecular Chaperones/genetics , Neurons/enzymology , Animals , Cell Line, Tumor , Cells, Cultured , Gene Knockdown Techniques , Humans , Mice , Mice, Transgenic , Molecular Chaperones/metabolism , Neurons/pathology , RNA, Small Interfering/pharmacologyABSTRACT
Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. Herein, is described a series of potent inhibitors based on an hydroxyethylamine (HEA) transition state mimetic template. These inhibitors interact with the non prime side of the enzyme using a novel edge-to-face interaction with Arg-296.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Arginine/chemistry , Ethylamines/chemistry , Protease Inhibitors/chemistry , Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases/chemistry , Amyloid Precursor Protein Secretases/metabolism , Animals , Binding Sites , Computer Simulation , Crystallography, X-Ray , Ethylamines/chemical synthesis , Ethylamines/therapeutic use , Protease Inhibitors/chemical synthesis , Protease Inhibitors/therapeutic use , Rats , Structure-Activity RelationshipABSTRACT
Epidemiological studies have shown an association between statin use and a decreased risk of dementia. However, the mechanism by which this beneficial effect is brought about is unclear. In the context of Alzheimer's disease, at least three possibilities have been studied; reduction in amyloid beta peptide (Abeta) production, the promotion of alpha-secretase cleavage and positive effects on neurite outgrowth. By investigating the effects of mevalonate pathway blockade on neurite outgrowth using real-time imaging, we found that rather than promote the production of neurite extensions, inhibition rapidly induced cell rounding. Crucially, neurite-like structures were generated through the persistence of cell-cell and cell-substrate adhesions and not through a mechanism of positive outgrowth. This effect can be strikingly enhanced by the over-expression of human amyloid precursor protein and is isoprenoid rather than cholesterol dependent.
Subject(s)
Amyloid beta-Protein Precursor/physiology , Mevalonic Acid/antagonists & inhibitors , Neurites/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Line, Tumor , Cell Shape/physiology , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Mevalonic Acid/metabolism , Microscopy, Video/methods , Neurites/drug effects , Neurites/pathology , Neuroblastoma/metabolism , Neuroblastoma/pathology , Neurogenesis/drug effects , Neurogenesis/physiologyABSTRACT
Our first generation of hydroxyethylamine transition-state mimetic BACE-1 inhibitors allowed us to validate BACE-1 as a key target for Alzheimer's disease by demonstrating amyloid lowering in an animal model, albeit at rather high doses. Finding a molecule from this series which was active at lower oral doses proved elusive and demonstrated the need to find a novel series of inhibitors with improved pharmacokinetics. This Letter describes the discovery of such inhibitors.
Subject(s)
Aspartic Acid Endopeptidases/antagonists & inhibitors , Ethylamines/chemistry , Protease Inhibitors/chemistry , Administration, Oral , Alzheimer Disease/drug therapy , Amyloid beta-Peptides/metabolism , Animals , Aspartic Acid Endopeptidases/metabolism , Binding Sites , Computer Simulation , Crystallography, X-Ray , Ethylamines/chemical synthesis , Ethylamines/pharmacology , Humans , Mice , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Rats , Structure-Activity Relationship , Thiazines/chemistry , Thiazines/pharmacologyABSTRACT
Our first generation of hydroxyethylamine BACE-1 inhibitors proved unlikely to provide molecules that would lower amyloid in an animal model at low oral doses. This observation led us to the discovery of a second generation of inhibitors having nanomolar activity in a cell-based assay and with the potential for improved pharmacokinetic profiles. In this Letter, we describe our successful strategy for the optimization of oral bioavailability and also give insights into the design of compounds with the potential for improved brain penetration.
Subject(s)
Aspartic Acid Endopeptidases/antagonists & inhibitors , Ethylamines/chemistry , Protease Inhibitors/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Amyloid beta-Peptides/metabolism , Animals , Aspartic Acid Endopeptidases/metabolism , Biological Availability , Dogs , Ethylamines/chemical synthesis , Ethylamines/pharmacokinetics , Mice , Mice, Knockout , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacokinetics , Rats , Structure-Activity RelationshipABSTRACT
Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. We have recently disclosed a series of transition-state mimetic BACE-1 inhibitors showing nanomolar potency in cell-based assays. Amongst them, GSK188909 (compound 2) had favorable pharmacokinetics and was the first orally bioavailable inhibitor reported to demonstrate brain amyloid lowering in an animal model. In this Letter, we describe the reasons that led us to favor a second generation of inhibitors for further in vivo studies.
Subject(s)
Aspartic Acid Endopeptidases/antagonists & inhibitors , Protease Inhibitors/chemistry , Thiazines/chemistry , Administration, Oral , Alzheimer Disease/drug therapy , Amyloid beta-Peptides/metabolism , Animals , Aspartic Acid Endopeptidases/metabolism , Binding Sites , Computer Simulation , Ethylamines/chemical synthesis , Ethylamines/chemistry , Ethylamines/pharmacology , Humans , Mice , Protease Inhibitors/pharmacokinetics , Rats , Structure-Activity Relationship , Thiazines/chemical synthesis , Thiazines/pharmacokineticsABSTRACT
BACE-1 inhibition has the potential to provide a disease-modifying therapy for the treatment of Alzheimer's disease. Optimization of a first generation of BACE-1 inhibitors led to the discovery of novel hydroxyethylamines (HEAs) bearing a tricyclic nonprime side. These derivatives have nanomolar cell potency and are orally bioavailable.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Benzothiadiazines/chemical synthesis , Cyclic S-Oxides/chemical synthesis , Ethylamines/chemical synthesis , Administration, Oral , Animals , Benzothiadiazines/pharmacokinetics , Benzothiadiazines/pharmacology , Biological Availability , Cyclic S-Oxides/pharmacokinetics , Cyclic S-Oxides/pharmacology , Dogs , Ethylamines/pharmacokinetics , Ethylamines/pharmacology , Models, Molecular , Rats , Structure-Activity RelationshipABSTRACT
This paper describes the discovery of non-peptidic, potent, and selective hydroxy ethylamine (HEA) inhibitors of BACE-1 by replacement of the prime side of a lead di-amide 2. Inhibitors with nanosmolar potency and high selectivity were identified. Depending on the nature of the P(1)(') and P(2)(') substituents, two different binding modes were observed in X-ray co-crystal structures.
Subject(s)
Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Combinatorial Chemistry Techniques , Ethylamines/chemical synthesis , Ethylamines/pharmacology , Alzheimer Disease/drug therapy , Amyloid beta-Protein Precursor/antagonists & inhibitors , Crystallography, X-Ray , Ethylamines/chemistry , Humans , Molecular Structure , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. Herein, is described the lead generation effort which resulted, with the support of X-ray crystallography, in the discovery of potent inhibitors based on a hydroxy ethylamine (HEA) transition-state mimetic. These inhibitors were capable of lowering amyloid production in a cell-based assay.
Subject(s)
Alzheimer Disease/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Combinatorial Chemistry Techniques , Ethylamines/chemical synthesis , Ethylamines/pharmacology , Alzheimer Disease/drug therapy , Amyloid beta-Protein Precursor/antagonists & inhibitors , Crystallography, X-Ray , Ethylamines/chemistry , Molecular Structure , Structure-Activity RelationshipABSTRACT
This article is focusing on further optimization of previously described hydroxy ethylamine (HEA) BACE-1 inhibitors obtained from a focused library with the support of X-ray crystallography. Optimization of the non-prime side of our inhibitors and introduction of a 6-membered sultam substituent binding to Asn-294 as well as a fluorine in the C-2 position led to derivatives with nanomolar potency in cell-based assays.
Subject(s)
Alzheimer Disease/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Combinatorial Chemistry Techniques , Ethylamines/chemical synthesis , Ethylamines/pharmacology , Alzheimer Disease/drug therapy , Amyloid beta-Protein Precursor/antagonists & inhibitors , Animals , Asparagine/chemistry , Crystallography, X-Ray , Disease Models, Animal , Ethylamines/chemistry , Fluorine/chemistry , Mice , Molecular Structure , Nanotechnology , Structure-Activity RelationshipABSTRACT
Generation and deposition of the amyloid beta (Abeta) peptide following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 and gamma-secretase is central to the aetiology of Alzheimer's disease. Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach for the treatment of Alzheimer's disease. We have designed a selective non-peptidic BACE-1 inhibitor, GSK188909, that potently inhibits beta-cleavage of APP and reduces levels of secreted and intracellular Abeta in SHSY5Y cells expressing APP. In addition, we demonstrate that this compound can effectively lower brain Abeta in vivo. In APP transgenic mice, acute oral administration of GSK188909 in the presence of a p-glycoprotein inhibitor to markedly enhance the exposure of GSK188909 in the brain decreases beta-cleavage of APP and results in a significant reduction in the level of Abeta40 and Abeta42 in the brain. Encouragingly, subchronic dosing of GSK188909 in the absence of a p-glycoprotein inhibitor also lowers brain Abeta. This pivotal first report of central Abeta lowering, following oral administration of a BACE-1 inhibitor, supports the development of BACE-1 inhibitors for the treatment of Alzheimer's disease.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/biosynthesis , Amyloid beta-Protein Precursor/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Brain/drug effects , Enzyme Inhibitors/pharmacology , Thiazines/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Administration, Oral , Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/chemistry , Animals , Aspartic Acid Endopeptidases/metabolism , Brain/metabolism , Brain/physiopathology , Cell Line, Tumor , Disease Models, Animal , Down-Regulation/drug effects , Down-Regulation/physiology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/therapeutic use , Humans , Male , Mice , Mice, Transgenic , Peptides/metabolism , Thiazines/chemical synthesis , Thiazines/therapeutic use , Treatment OutcomeABSTRACT
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse models show features that closely mimic those seen in the clinical situation, reflected in the molecular changes observed in mouse models and in tissues from patients. We report a dramatic increase in the expression of amyloid precursor protein (APP) in the hindlimb muscles, but not the spinal cord of the G93A transgenic mouse model, significantly before the appearance of clinical abnormalities. APP levels were unchanged in nontransgenic mice and in mice overexpressing human wild-type Cu/Zn-dependent superoxide dismutase 1 (SOD1). Preliminary results indicate a similar change in APP expression in human deltoid muscle samples from ALS patients compared with age-matched controls. The inhibitory role of APP in innervation at the neuromuscular junction and increased expression in inclusion-body myositis suggest that presymptomatic upregulation of APP may be consistent with a potential role for APP in ALS pathology.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Amyotrophic Lateral Sclerosis/metabolism , Muscle, Skeletal/metabolism , Adult , Aged , Amyloid Precursor Protein Secretases , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/physiology , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Animals , Aspartic Acid Endopeptidases , Disease Models, Animal , Endopeptidases/genetics , Endopeptidases/metabolism , Female , Hindlimb/metabolism , Humans , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Middle Aged , Muscle, Skeletal/physiopathology , Presenilin-1 , Spinal Cord/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase-1 , Up-RegulationABSTRACT
Genetic analysis of familial Alzheimer's disease has revealed that mutations in the gamma-secretase enzyme presenilin promote toxic Abeta secretion; however, presenilin mutations might also influence tau hyperphosphorylation and neurodegeneration through gamma-secretase-independent mechanisms. To address this possibility and determine whether other components of the gamma-secretase complex possess similar regulatory functions, we analyzed the roles of presenilin, nicastrin, and aph-1 in a Drosophila model for tau-induced neurodegeneration. Here, we show that presenilin and nicastrin prevent tau toxicity by modulating the PI3K/Akt/GSK3beta phosphorylation pathway, whereas aph-1 regulates aPKC/PAR-1 activities. Moreover, we found that these transmembrane proteins differentially regulate the intracellular localization of GSK3beta and aPKC at cell junctions. Inhibition of gamma-secretase activity neither interfered with these kinase pathways nor induced aberrant tau phosphorylation. These results establish new in vivo molecular functions for the three components of the gamma-secretase complex and reveal a different mechanism that might contribute to neuronal degeneration in Alzheimer's disease.
Subject(s)
Drosophila Proteins/metabolism , Endopeptidases/metabolism , Intercellular Junctions/metabolism , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , tau Proteins/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Amyloid Precursor Protein Secretases , Animals , Animals, Genetically Modified , Down-Regulation/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Endopeptidases/drug effects , Endopeptidases/genetics , Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Intercellular Junctions/genetics , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , Mutation/genetics , Nerve Degeneration/genetics , Nerve Degeneration/metabolism , Nerve Degeneration/physiopathology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Presenilin-1 , Protein Kinase C/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Signal Transduction/physiology , tau Proteins/genetics , tau Proteins/toxicityABSTRACT
Apolipoprotein (apo) E4 is a risk factor for Alzheimer's disease (AD) and other neurodegenerative diseases, compared to wild-type apoE3. The mechanism(s) is unknown. One possibility, demonstrated in peripheral tissue cell lines, is that apoE stimulates nitric oxide synthase (NOS) via a receptor-dependent signalling pathway and that apoE4 generates inappropriate amounts of nitric oxide (NO) compared to apoE3. Prior to biochemical investigations, we have quantified the expression of several candidate receptor genes, including low-density lipoprotein-receptor (LDL-r) family members and scavenger receptor class B, types I and II (SR-BI/II), as well as the three NOS isoenzymes and protein kinase B (Akt), in 38 human cell lines, of which 12 derive from brain. Expression of apoE receptor 2 (apoER2), a known signalling receptor in brain, was readily detected in SH-SY-5Y and CCF-STTG1 cells, common models of neurons and astrocytes, respectively, and was highest in H4 neuroglioma, NT-2 precursor cells and IMR-32 neuroblastoma cells. Transcripts of the other lipoprotein receptors were widely, but variably, distributed across the different cell types. Of particular note was the predominant expression of SR-BII over SR-BI in many of the brain-derived cells. As the C-terminus of SR-BII, like apoER2, contains potential SH3 signalling motifs, we suggest that in brain SR-BII functions as a signal transducer receptor.
Subject(s)
Astrocytes/metabolism , Brain/metabolism , Gene Expression Profiling/methods , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , Neurons/metabolism , Receptors, Immunologic/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Apolipoproteins E/metabolism , CD36 Antigens , Cell Line , Humans , Nitric Oxide/metabolism , Receptors, Scavenger , Scavenger Receptors, Class BABSTRACT
The amyloidogenic pathway leading to the production and deposition of Abeta peptides, major constituents of Alzheimer disease senile plaques, is linked to neuronal metal homeostasis. The amyloid precursor protein binds copper and zinc in its extracellular domain, and the Abeta peptides also bind copper, zinc, and iron. The first step in the generation of Abeta is cleavage of amyloid precursor protein by the aspartic protease BACE1. Here we show that BACE1 interacts with CCS (the copper chaperone for superoxide dismutase-1 (SOD1)) through domain I and the proteins co-immunoprecipitate from rat brain extracts. We have also been able to visualize the co-transport of membranous BACE1 and soluble CCS through axons. BACE1 expression reduces the activity of SOD1 in cells consistent with direct competition for available CCS as overexpression of CCS restores SOD1 activity. Finally, we demonstrate that the twenty-four residue C-terminal domain of BACE1 binds a single Cu(I) atom with high affinity through cysteine residues.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Copper/metabolism , Cytoplasm/enzymology , Molecular Chaperones/metabolism , Superoxide Dismutase/metabolism , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases/genetics , Binding Sites , Cytoplasm/genetics , Endopeptidases , Humans , Molecular Chaperones/biosynthesis , Molecular Chaperones/genetics , Protein Structure, Tertiary , RatsABSTRACT
Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimer's disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its beta-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1-APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/biosynthesis , Cell Membrane/metabolism , Cholesterol/metabolism , Hippocampus/metabolism , Neurons/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/physiopathology , Amyloid Precursor Protein Secretases , Amyloid beta-Protein Precursor/metabolism , Animals , Aspartic Acid Endopeptidases/metabolism , Biomarkers/metabolism , Cell Compartmentation/physiology , Cells, Cultured , Cholesterol/deficiency , Endopeptidases , Hippocampus/cytology , Hippocampus/physiopathology , Humans , Membrane Microdomains/metabolism , Mice , Mice, Transgenic , Neurons/cytology , Rats , Subcellular Fractions/metabolism , Thy-1 Antigens/metabolism , Up-Regulation/physiologyABSTRACT
Neurofilament middle and heavy chains (NFM and NFH) are heavily phosphorylated on their carboxy-terminal side-arm domains in axons. The mechanisms that regulate this phosphorylation are complex. Here, we demonstrate that p38alpha, a member of the stress-activated protein kinase family, will phosphorylate NFM and NFH on their side-arm domains. Aberrant accumulations of neurofilaments containing phosphorylated NFM and NFH side-arms are a pathological feature of amyotrophic lateral sclerosis (ALS) and we also demonstrate that p38alpha and active forms of p38 family kinases are associated with these accumulations. This is the case for sporadic and familial forms of ALS and also in a transgenic mouse model of ALS caused by expression of mutant superoxide dismutase-1 (SOD1). Thus, p38 kinases may contribute to the aberrant phosphorylation of NFM and NFH side-arms in ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/enzymology , Mitogen-Activated Protein Kinases/metabolism , Motor Neurons/enzymology , Nerve Degeneration/enzymology , Neurofilament Proteins/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , COS Cells , Disease Models, Animal , Fetus , Mice , Mice, Transgenic , Mitogen-Activated Protein Kinase 14 , Motor Neurons/pathology , Mutation/genetics , Nerve Degeneration/genetics , Nerve Degeneration/physiopathology , Phosphorylation , Rats , Superoxide Dismutase/deficiency , Superoxide Dismutase/genetics , Superoxide Dismutase-1 , p38 Mitogen-Activated Protein KinasesABSTRACT
Caspases play a key role during apoptotic execution. In an attempt to elucidate the specific role of caspase-7 we generated a chicken DT40 cell line in which both alleles of the gene were disrupted. Viability assays showed that caspase-7-/- clones are more resistant to the common apoptosis-inducing drugs etoposide and staurosporine. Caspase-7-/- cells show a delay in phosphatidylserine externalization and DNA fragmentation as well as cleavage of the caspase substrates poly(ADP-ribose) polymerase 1 and lamins B1 and B2. Caspase affinity labeling and activity assays indicated that deficient cells exhibit a delay in caspase activation compared with wild type DT40 cells, providing an explanation for the differences in apoptotic execution between caspase-7 null and wild type DT40 cells. These results strongly suggest that caspase-7 is involved earlier than other effector caspases in the apoptotic execution process in DT40 B lymphocytes.